CH 3 CH 3 CH 3 CH 3 O O CH 3 C H 3 C H 3 CH 3 CH 3 C H 3 O O NH 2 N H 2 O O NH 2 NH 2 O O O CH 3 CH 3 CH 3 CH 3 O O OH O H O H O O OH O H OH O H O OH OH OH O O O H OH OH OH EXCITED-STATE PROPERTIES OF HYDROPHILIC CAROTENOIDS Pavel Chábera 1 , K. Razi Naqvi 2 , Thor Bernt Melø 2 , Hans-Richard Sliwka 3 , Vassilia Partali 3 , Sam Lockwood 4 , H.L. Jackson 5 , Tomáš Polívka 1,6 1) Institute of physical Biology, Zamek 136, 373 33 Nove Hrady, Czech Republic 2) Department of Physics, Norwegian University of Science and Technology (NTNU),N-7491, Trondheim, Norway 3) Department of Chemistry, NTNU, N-7491 Trondheim, Norway 4) NELHA, 73-4460 Queen Kaahumanu Hwy,Kailua-Kona, Hawaii 96740 5) Cardax Pharmaceuticals, 99-193 Aiea Heights Drive, Suite 400, Aiea, Hawaii 96701 6) Institute of Plant Molecular Biology, Biological Centre, Czech Academy of Sciences 350 400 450 500 550 600 0.0 0.2 0.4 0.6 0.8 1.0 30000 28000 26000 24000 22000 20000 18000 16000 Wavenumber (cm −1 ) ΔA (a.u.) Wavelength (nm) Crocin H 2 O Crocin MetOH 500 550 600 650 700 0.0 0.5 1.0 -0.5 0.0 0.5 1.0 21000 20000 19000 18000 17000 16000 15000 14000 Energy (cm -1 ) Wavelength (nm) Crocin H 2 O Crocin MetOH ΔA (a.u.) Astalysine H 2 0 Astalysine MetOH Astaxanthin MetOH -2 0 2 4 6 8 10 12 14 16 18 20 22 0.0 0.2 0.4 0.6 0.8 1.0 ΔA (a.u.) Time (ps) Astalysine H 2 O @ 642 nm Astalysine MetOH @ 631 nm Astaxanthin MetOH @ 635 nm 4.5 ps 2.2 ps 4.9 ps (1) Gainer, J.L; Grabiak, R.C., 2008, US 7351844. (2) Foss, B.J.; Nadolski, G.; Lockwood, S.F., Mini-Rev. Med. Chem. 2006, 6, 953–969. (3) Zigmantas, D.; Hiller, R.G.; Sharples, F.P.; Frank, H.A.; Sundström, V.; Polivka, T., Phys. Chem. Chem. Phys. 2004, 6, 3009–3016. (4) Ilagan, R.P.; Christensen, R.L.; Chapp, T.W.; Gibson, G.N.; Pascher, T.; Polivka, T.; Frank, H.A. J. Phys. Chem. A 2005, 109, 3120–3127. Crocin Astalysine 0 50 100 150 200 250 300 350 400 450 500 0.0 0.2 0.4 0.6 0.8 1.0 135.5 ps 61 ps ΔA (a.u.) Time (ps) Crocin H2O @ 528 nm Crocin MetOH @ 518 nm 350 400 450 500 550 600 0.0 0.2 0.4 0.6 0.8 1.0 30000 28000 26000 24000 22000 20000 18000 16000 Energy (cm −1 ) Wavelength (nm) ΔA (a.u.) Astalysine H 2 O Aslalysine MetOH Astaxanthin MetOH Introduction: Carotenoids represent a group of natural pigments that are inherently hydrophobic, but a few natural water-soluble examples are known. Moreover, a hydrophilic derivatives of natural carotenoids have been synthesized recently (1,2). Here we present a study of excited-state properties of two water- soluble carotenoids, crocin and astalysine. Crocin is a natural chemical compound found in flower crocus (Crocus longiflorus) where is located in the carpels, and is responsible for typical red-gold colour of saffron. Astaxanthin-lysine (astalysine) is an artificial compound derived from a carotenoid astaxanthin and a-amino acid lysine. Since both contain conjugated carbonyl groups, their hydrophilicity opens the possibility to study polarity-induced effects attributed to the conjugated carbonyl group (3) in water, a solvent with extreme polarity. Materials & Methods: Time-resolved transient absorption spectra were collected by a commercial femtosecond amplifier (Integra-i , Quantronix) producing ~140 fs pulses centered at 790 nm and operating at repetition rate of 1 KHz. The amplifier output was divided into two beams. One was used to pump an optical parametrical amplifier (Topas, Light Conversion) to provide excitation pulses, the other was used to generate a white-light continuum in a 1 mm sapphire plate which served as a probe beam. The probe was further split into probe and reference beams detected by double diode-array coupled with spectrograph. STEADY STATE ABSORPTION SPECTRA of crocin (left) and a astalysine (right) in H 2 O (black line) and in methanol (red line). Absorption spectrum of astaxanthin in methanol is also showed (green line). All spectra were measured at room temperature and are normalized to the absorption maximum. The absorption spectrum of crocin, which has 7 conjugated C=C and two conjugated C=O bonds, exhibits well-resolved vibrational bands in methanol, but the vibrational structure is blurred in H 2 O. This behaviour was observed in carbonyl carotenoids upon changing the solvent from nonpolar to polar (3). Yet, crocin exhibits well-resolved vibrational bands even in polar methanol, and the characteristic polarity-induced effect is observed only in H 2 O. In astalysine, only a slight red shift and spectral broadening is observable in H 2 O as compared to methanol. TRANSIENT ABSORPTION SPECTRA of astalysine (upper panel) and crocin (lower panel) in H 2 O (black) and in methanol (red). Astaxanthin in methanol is also shown (green). Transient spectra were recorded at room temperature 1 ps after excitation. All spectra are normalized to maximum. Transient spectrum of astalysine (conjugation length N=13, 11 C=C bonds and 2 conjugated C=O groups) in methanol is very similar to that one of astaxanthin in methanol, indicating that the two attached lysine groups do not alter the excited-state properties. Even though the S 1 lifetimes of astalysine and crocin (conjugated length N=9, 7 C=C bonds and 2 conjugated C=O groups) in H 2 O is significantly shorter as compared with those in methanol (see table), no bands attributable to the presence of an intramolecular charge transfer state (ICT) have been found in the transient absorption spectra in H 2 O. KINETICS measured at the maximum of the S 1 -S n transient absorption band following excitation to the lowest vibrational band of S 2 state. Kinetics were measured in methanol and H 2 O for crocin (left panel) and for astalysine (right panel - together with astaxanthin in methanol). Kinetics are normalized to maximum, solid lines correspond to fits. Kinetics show clear shortening of the S 1 lifetime of both carotenoids upon change of solvent from methanol to H 2 O. For crocin, the S 1 lifetime is shortened from 135 ps in methanol to 61 ps in H 2 O. Similar polarity-induced shortening, from 4.5 to 2.2 ps, is observed in astalysine. While no polarity-induced effects on the S 1 lifetime were found for astaxanthin in a series of solvents with different polarities (4), it is obvious that the large polarity of H 2 O can shorten the S 1 lifetime, an effect observed earlier only in carbonyl carotenoids with fewer than 10 conjugated double bonds. The S 1 -S N signal of astaxanthin in methanol decays with 4.9 ps time constant indicating that the two lysine groups of astalysine do not alter the excited-state properties. CONCLUSIONS • The polarity-induced shortening of S 1 lifetime is observed in both crocin and astalysine • Characteristic loss of vibrational structure is observed in absorption spectrum of crocin • No signs of ICT-like bands were observed in transient absorption spectra • Polarity-induced S 1 lifetime shortening can be observed even for carbonyl carotenoids with conjugation length higher than 10 if high polarity solvent is used (i.e. H 2 O) Carotenoid Solvent S 0 -S 2 max (cm -1 / nm) S 1 -S N max (cm -1 / nm) Excitation λ (nm) τ S1 (ps) Crocin Methanol 23150 / 432 19230 / 520 465 135 Crocin H 2 O 22620 / 442 19480 / 525 475 61 Astalys ine Methanol 22790 / 481 16000 / 625 510 4.5 Astalys ine H 2 O 20530 / 487 15700 / 637 520 2.2 Astaxanthin Methanol 21050 / 475 16530 / 605 505 4.9